The enormous increase in the demand for synthetic oligonucleotides fueled by the advances in DNA technology over the last few decades has been accelerated by recent progress in sequencing and decoding whole genomes, particularly the human genome. A number of methods in molecular biology and DNA-based diagnostics to amplify, detect, analyze and quantify nucleic acids are dependent on chemically synthesized oligonucleotides which are employed as primers and probes to amplify or to detect nucleic acid targets. Synthetic nucleic acids are also employed in therapeutic applications to block the expression of specific genes in a sequence dependent manner or to stimulate the immune system and present a very promising class of highly specific novel therapeutic agents which have the potential to be designed based on their sequence.
The properties of synthetic oligonucleotides can be manipulated and fine-tuned to the demands of their particular application by chemically conjugating the oligonucleotides to a variety of modifiers. Examples of modifiers include reporter groups to allow the facile detection of the modified oligonucleotides, e.g. fluorescent dyes, haptens to facilitate the specific capture and detection of oligonucleotides and their reaction products in diagnostic assays, e.g. biotin or digoxigenin, lipophilic modifiers to enhance the uptake of oligonucleotides in cells, e.g. cholesterol, modifiers to increase the biocompatibility and to reduce the exonucleolytic degradation of oligonucleotides, e.g. polyethylene or other groups which block the terminal hydroxyl groups, affinity modifiers to increase the affinity of oligonucleotides to complementary sequences, e.g. intercalators or nucleic acid groove binders, and peptides to achieve a variety of specific effects including targeted delivery to specific cell lines in an organism.
The development of efficient methods for the chemical synthesis of oligonucleotides and their conjugates over the past two decades has facilitated the routine provision of oligonucleotides of defined sequence and modifications. The current state of the art in oligonucleotide synthesis is automated solid phase synthesis using phosphoramidite chemistry, which in particular is based on the developments of McBride et al. (1983) Tetrahedron Letters 24:245-248 and Sinha et al. (1983) Tetrahedron Letters 24:5843-5846. These methods, together with related methods such as the hydrogen-phosphonate chemistry, have been extensively reviewed by Beaucage et al. (1992) Tetrahedron 48:2223-2311. Each of these references is specifically incorporated herein by reference in its entirety. The conjugation of oligonucleotides can be achieved through the incorporation of reagents in the solid phase synthesis which either introduce a functional group to the oligonucleotides for further selective manipulations or which directly introduce the desired modification in the course of the solid phase synthesis, as reviewed by Grimm et al. (2000) Nucleosides, Nucleotides & Nucleic Acids 19:1943-1965, and Beaucage et al. (1993) Tetrahedron 49:1925-1963, each of which is specifically incorporated herein by reference in its entirety.
The incorporation of modifications in synthetic oligonucleotides at their 3′-terminus has gained particular attention because a 3′-modification, in contrast to the more commonly applied 5′-modification, leaves the 5′-terminus of an oligonucleotide available for further synthetic or enzymatic modification and provides considerable stabilization against degradation in biological fluids. Unmodified nucleic acids are degraded in biological fluids, e.g. in cultured cells or whole organisms, by nucleases. 3′-Exonuclease activities contribute greatly to the observed instability. 3′-Terminal modifications provide significant stabilization against enzymatic degradation, as demonstrated e.g. for 3′-phosphopropyl amine oligonucleotides by Zendegui et al. (1992) Nucleic Acids. Res. 20:307-314, which is incorporated herein by reference in its entirety. 3′-Terminal modifications are therefore suitable to enhance the in-vivo stability of nucleic acids based therapeutics, such as antisense oligonucleotides, small interfering RNA, synthetic ribozymes and aptamers. The 3′-specific attachment of modifier groups, such as lipophilic groups, intercalating agents, reporter groups, polyethylene glycols, small peptides and other groups may further enhance the nucleolytic stability of oligonucleotides. These groups may also facilitate their penetration of cell membranes, increase their affinity to complementary target nucleic acids, or make them traceable in a biological system. An example of the application of 3′-amino modifications in the field of oligonucleotide based therapeutics is provided by Zerial et al. (1987) Nucleic Acids. Res. 15:9909-9919, which is incorporated herein by reference in its entirety.
The 3′-modification of oligonucleotides is particularly useful in the synthesis of bi-fluorescent probes which contain two different fluorescent dyes at their 3′- and 5′-termini. Bi-fluorescent probes are widely employed in oligonucleotide based diagnostic assays such as real-time quantitative PCR. Bi-fluorescent probes are also employed as molecular beacons; see Tyagi et al. (1996) Nat. Biotechnol. 14:303-308, hydrolysis probes (Taqman™ technology, Perkin-Elmer Applied Biosystems, Foster City, Calif., USA), see Heid et al. (1996) Genome Research 6:986-994, and scorpion probes, see Whitcombe et al. (1999) Nat. Biotechnol. 17:804-807, each of which is incorporated herein by reference in its entirety.
The incorporation of a primary amino group at the 3′-terminus of a synthetic oligonucleotide is of particular interest due to the high reactivity of primary amino groups which allows for the chemoselective derivatization of the corresponding oligonucleotides. 3′-Amino oligonucleotides can be conveniently conjugated to a variety of reporters, haptens or other modifiers by reacting the amino group in a selective manner with active ester derivatives of the moieties to be conjugated. Many active ester derivatives of such moieties are either commercially available or can be synthesized by standard esterification reactions. 3′-Amino oligonucleotides can also be covalently attached to surfaces with electrophilic groups, as described e.g. by Gerry et al. (1999) J. Mol. Biol. 292:251-262, which is incorporated herein by reference in its entirety.
3′-Amino oligonucleotides are conveniently prepared using solid phase synthetic methods on specialty solid supports. Such supports are applied in the same manner as conventional supports for the synthesis of unmodified oligonucleotides, but release oligomers with free 3′-amino groups during the standard deprotection of the oligonucleotides. A suitable support for the synthesis of 3′-amino derivatized oligonucleotides would ideally fulfill the following criteria:                A) it would be compatible with and stable under the standard phosphoramidite synthetic method for oligonucleotides;        B) it would comprise a linkage to the oligonucleotide that is cleaved during the deprotection of the nucleobases, wherein said cleavage does not require the introduction of reagents which are not commonly employed in the deprotection of oligonucleotides;        C) it would be cleavable from the oligonucleotide in a reaction time that is comparable to the time employed in standard deprotection conditions for the removal of base protective groups;        D) it would provide the 3′-amino oligonucleotide without side products derived from modifications of the amino group, e.g. acylations of the amine;        E) it would not generate diasteromeric mixtures of oligonucleotides due to the presence of chiral centers on the support; and        F) it would be preparable in a simple and efficient manner.Standard deprotection conditions are such conditions that are commonly employed to remove the base protective groups: isobutyryl from guanine residues and benzoyl from adenine and cytosine residues, e.g. an incubation of the support in concentrated aqueous ammonia at 55° C. for 8 hours.        
Several reports on derivatized solid supports that are suitable for the synthesis of oligonucleotides with 3′-amino modifications have already appeared in the literature, but none of the described products meet all of the above criteria for a generally useful support. The known supports can be divided in two groups. The first group contains a protected amino group wherein the protective group is attached to the support and the 3′-amino oligonucleotide is released upon the cleavage of the protective group. The protective group for the amino function also serves as a linker in this group of supports, which connects the solid phase of the support with the oligonucleotide. The second group of supports contains a branched linker wherein the linker contains a protected amino group on a side arm and the oligonucleotide is attached to the solid phase of the support through another functionality of the linker. In this group of supports, the 3′-amino oligonucleotide is released upon cleavage of the linker from the support and the amino function is liberated either simultaneously or in a separate step through the removal of the side arm protective group.
An example out of the first group of supports for the synthesis of 3′-amino oligonucleotides has been described by Asseline et al. (1990) Tetrahedron Letters 31:81-84, which is incorporated herein by reference in its entirety. The linker of the support described by Asseline et al. contains a disulfide group, which is cleaved with dithiothreitol to release the 3′-amino oligonucleotide. Dithiothreitol is not commonly employed in the solid phase synthesis of oligonucleotides and its application is undesirable. Additionally, the described support is not easy to prepare and requires multiple synthetic steps in solution and on the support.
Other examples of the first group of solid supports have been described by Kumar et al. (1996) Bioorg. Med. Chem. Lett. 6:2247-2252, which is incorporated herein by reference in its entirety. The linkers employed in the supports described by Kumar et al. contain a sulfonylethyl group that is cleaved in concentrated aqueous ammonia at 55° C. to release the 3′-amino oligonucleotide products. The described incubation time in concentrated ammonia is 16 hours, which exceeds the standard deprotection time for oligonucleotides. Additionally, supports are prepared in multi-step processes and a variety of reagents are employed to manipulate the functional groups of the corresponding solid phase intermediates. Such reactions are difficult to monitor and the purity of the intermediates can not easily be demonstrated. The supports are therefore difficult to prepare and their use is not compatible with standard deprotection conditions and with base sensitive modifications of oligonucleotides.
In another example of the first group of supports Petrie et al. (1992) Bioconjugate Chem. 3:85-87, and Reed et al., U.S. Pat. No. 5,419,966, each of which is incorporated herein by reference in its entirety, describe the application of the phthaloyl protective group. The phthaloyl group is removed from the 3′-amine oligonucleotide with concentrated ammonia in 16 hours at 55° C. The required time for the removal of the phthaloyl protective group is longer than the standard deprotection time for oligonucleotides, which reduces the throughput in the synthesis of oligonucleotides and makes the support incompatible with base-sensitive modifications. A similar support is also disclosed by Lyttle et al. (1997) Bioconjugate Chem. 8:193-198, which is incorporated herein by reference in its entirety. Lyttle et al. describe a linker based on trimellitic acid that is cleavable from the 3′-amino oligonucleotide by treatment with concentrated ammonia at 55° C. for 18-24 hours. The applied reaction time for the cleavage of the support from the oligonucleotide is longer than the standard reaction time for the deprotection of nucleobases. In addition, 20-30% of a side product was observed in the synthesis of a 3′-amino 14-mer oligonucleotide, which was tentatively characterized as the 3′-amino oligonucleotide conjugated to trimellitic acid at the amino group, which indicates incomplete cleavage between the trimellitic acid linker and the oligonucleotide even under the prolonged time of reaction in ammonia.
In other variations of supports which utilize an amino protective group as part of the linker to the support Avino et al. (1996) Bioorg. Med. Chem. 4:1649-1658, which is incorporated herein by reference in its entirety, applied derivatized o-nitrophenylethyl-(o-NPE) and 9-fluorenylmethyloxycarbonyl (Fmoc) amino protective groups attached to the support through substituents at their aromatic rings. The linker based on the Fmoc-group was, however, believed not to be stable enough under the standard conditions of phosphoramidite mediated oligonucleotide synthesis as low yields of 3′-amino oligonucleotides were observed. In contrast, the o-NPE-group could not be cleaved completely with concentrated ammonia and the stronger base DBU had to be used as a 0.5 M solution in pyridine for 16 hours to achieve efficient cleavage. The application of a solution of DBU in pyridine is an additional step, which requires additional work-up steps and is therefore undesirable.
Examples of the application of a branched linker based on an 3-amino-1,2-propanediol linker unit are provided by Nelson et al. (1989) Nucleic Acids Res. 17:7187-7194, and U.S. Pat. No. 5,141,813, each of which is incorporated herein by reference in its entirety. The linker in the solid support of Nelson et al. utilizes the vicinal hydroxyl groups of 3-amino-propane-1,2-diol as attachment points for the oligonucleotide and the solid phase whereas the amino group of the linker is Fmoc protected. Oligonucleotide products synthesized on this support contain a 3′-amino-2-hydroxypropylphosphate moiety. The support has several disadvantages. The Fmoc protective group of the amino function is not completely stable to the conditions applied in a phosphoramidite mediated oligonucleotide synthesis and is partially removed in the process. The resulting free amino group is exposed to the capping reagent acetic anhydride during the oligonucleotide synthesis and the amino groups are therefore partially acetylated. The acetylated amino groups are stable during the deprotection of the nucleoside bases and the 3′-amino oligonucleotide is therefore contaminated with the corresponding 3′-acetylamino species. The utilization of vicinal diol groups also facilitates the cleavage of the 3-amino-2-hydroxypropylphosphate moiety from the oligonucleotide products through cyclic phosphate intermediates, resulting in unmodified 3′-OH oligonucleotides. 3′-acetylamino oligonucleotides and non-modified 3′-OH oligonucleotides were observed as contaminants in 3′-amino oligonucleotides prepared with the support of Nelson et al. e.g. by Vu et al. (1995) Bioconjugate Chem. 6:599-607, which is incorporated herein by reference in its entirety. Another disadvantage of solid supports based on the branched 3-amino-propane-1,2-diol element is the introduction of a chiral center to the 3′-amino oligonucleotide products. Oligonucleotides prepared on this support exist as mixtures of two diastereoisomers as a consequence of the undefined stereochemistry of the carbon atom at the linker branching point, i.e. the carbon atom in the 2-position of the 3-amino-propane-1,2-diol skeleton. The existence of oligonucleotide diastereoisomers complicates the analysis of the 3′-amino oligonucleotides, as well as, their subsequent application in the conjugation of reporter molecules or haptens.
Nelson et al. also describe a solid support based on the branched linker unit 2-(4-aminobutyl)-propane-1,3-diol (i.e. 6-amino-2-hydroxymethyl-hexan-1-ol), see Nelson et al., U.S. Pat. No. 5,942,610, which is incorporated herein by reference in its entirety. This solid support is an improved version of the support based on the 3-amino-1,2-propanediol linker unit. It overcomes some of the associated disadvantages, because it utilizes a 1,3-diol system for the attachment of the oligonucleotide and the solid phase to the linker in contrast to the vicinal 1,2-diol system that was employed in the prior support. The undesired formation of unmodified 3′-OH oligonucleotides through cyclic phosphate intermediates is effectively suppressed in this system. The support, nevertheless, still has the following major disadvantages. It carries an Fmoc group to protect the amino function on the branch of the linker, which as noted above is not completely stable to the conditions employed in the capping steps of a phosphoramidite mediated oligonucleotide synthesis resulting in the formation of amino-acetylated side products. It also introduces a chiral center to the 3′-amino oligonucleotides, i.e. the carbon atom in the 2-position of the propane-1,3-diol system. This leads to two diastereomeric oligonucleotide products in each 3′-amino oligonucleotide, which as also noted above complicates the analysis of the oligomer and the monitoring of subsequent applications of the 3′-amino oligonucleotides.
Solid supports that contain an amino function on a branched linker can be further derivatized by conjugating the amino function to small molecules, haptens or reporter groups. The synthesis of oligonucleotides on such derivatized supports results in the corresponding 3′-modified oligonucleotides after cleavage and deprotection, which alleviates the need to prepare the corresponding 3′-modified oligonucleotides by post synthetic conjugation methods from 3′-amino oligonucleotides. The utility of this approach has been demonstrated e.g. by Gamper et al. (1993) Nucleic Acids Res. 21:145-150, with a 5-hydroxymethyl-pyrrolidine-(3R-trans)-3-ol linker element in the synthesis of oligonucleotides conjugated to acridine and to cholesterol at their 3′-end, and by Stetsenko et al. (2001) Bioconjugate Chem. 12:576-586, with a homoserine based linker element in the synthesis of oligonucleotides conjugated to 4-iodophenyl acetic acid, 6-carboxyfluorescein, biotin and other small molecules at their 3′-end, and by Mullah et al. (1998) Nucleic Acids Res. 26:1026-1031, with a 2-amino-propane-1,3-diol linker element in the synthesis of 3′-TAMRA modified oligonucleotides. Each of these references is specifically incorporated herein by reference in its entirety. Although this approach is useful for targeting a particular conjugate, it is limited in that a specialty support must be prepared individually for every hapten or reporter to be conjugated. In contrast, sequences with free amino groups, once prepared on a standard support, can be aliquoted and conjugated to a variety of different small molecules, haptens or reporter groups, thus eliminating the need to conduct multiple oligonucleotide synthesis if the same sequence is desired with different 3′-modifications. Additionally, oligonucleotides with free amino groups can be stored and used at a later date for the conjugation of another molecule.
The method of using specialty derivatized supports for each modification is also limited to those modifications that are stable under the conditions of oligonucleotide synthesis and under the conditions of cleavage and deprotection of the oligonucleotides. Many desirable 3′-modifications do not fulfill the stability criterion and can not be prepared with such solid supports. Other modifications require non-standard treatments or specialty reagents in the assembly of the oligonucleotide chain or in the cleavage and deprotection of the oligonucleotides as exemplified in the preparation of 3′-TAMRA modified oligonucleotides described by Mullah et al., wherein a mixture of tert-butylamine, methanol and water in a ratio of 1:1:2, v/v, is used to cleave and deprotect the conjugated oligonucleotide. Non-standard reagents are highly undesirable in routine schemes for the preparation of oligonucleotides, because modified and unmodified oligonucleotides are typically prepared in the same synthesis facilities by the same personnel and should be fully compatible with each other in order to obtain economic viability and to reduce the probability of errors resulting from the use of different reagents for different sets of oligonucleotides.
Although, as exemplified above, a variety of solid supports for the synthesis of 3′-amino oligonucleotides has been described, and some of the described solid supports are commercially available, there is no known solid support that combines all of the desired favorable features of such a support described in the criteria A) to F) above. The known supports either require extended cleavage and deprotection times, or inherently result in side products such as acetylated derivatives of the 3′-amino oligonucleotides, or result in diastereomeric mixtures of 3′-amino oligonucleotide products, or suffer from a combination of these disadvantages.
The present invention discloses novel methods and solid supports for the synthesis of 3′-amino oligonucleotides wherein the cleavage of the oligonucleotides from the support and the removal of their base protective groups can be conducted under mild alkaline conditions and wherein the 3′-amino oligonucleotides are obtained as single diastereoisomers free from side products. The novel solid supports described herein contain an ortho-hydroxymethyl benzoyl protective group (HMB-group) for the amino function wherein the hydroxymethyl group is employed for the attachment of the protective group to the solid phase of the support and the carbonyl group of the benzoyl moiety serves as the conjugation point for the amino-oligonucleotide. The HMB-group may optionally contain additional substituents in the aromatic ring.